Discovery of novel propargylamine-modified 4-aminoalkyl imidazole substituted pyrimidinylthiourea derivatives as multifunctional agents for the treatment of Alzheimer's disease

Accepted Manuscript Discovery of novel propargylamine-modified 4-aminoalkyl imidazole substituted pyrimidinylthiourea derivatives as multifunctional agents for the treatment of Alzheimer's disease Yi-xiang Xu, Huan Wang, Xiao-kang Li, Sheng-nan Dong, Wen-wen Liu, Qi Gong, Tian-duan-yi Wang, Yun Tang, Jin Zhu, Jian Li, Hai-yan Zhang, Fei Mao PII:

S0223-5234(17)30628-1

DOI:

10.1016/j.ejmech.2017.08.025

Reference:

EJMECH 9669

To appear in:

European Journal of Medicinal Chemistry

Received Date: 11 February 2017 Revised Date:

7 August 2017

Accepted Date: 8 August 2017

Please cite this article as: Y.-x. Xu, H. Wang, X.-k. Li, S.-n. Dong, W.-w. Liu, Q. Gong, T.-d.-y. Wang, Y. Tang, J. Zhu, J. Li, H.-y. Zhang, F. Mao, Discovery of novel propargylamine-modified 4aminoalkyl imidazole substituted pyrimidinylthiourea derivatives as multifunctional agents for the treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.08.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Discovery of novel propargylamine-modified 4-aminoalkyl imidazole substituted pyrimidinylthiourea derivatives as multifunctional agents for the treatment of Alzheimer’s Disease

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Yi-xiang Xua,1, Huan Wangb,c,1, Xiao-kang Lia, Sheng-nan Dongb, Wen-wen Liua, Qi Gongb, Tian-duan-yi Wanga, Yun Tanga, Jin Zhua, Jian Lia,*, Hai-yan Zhangb,*, Fei Maoa,* a

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

b

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University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

c

University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049,

China

*

These authors contributed equally to this work. To

whom

correspondence

should

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1

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Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China

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[email protected], [email protected].

be

addressed

[email protected],

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ABSTRACT: A series of novel propargylamine-modified pyrimidinylthiourea derivatives (1-3) were designed and synthesized as multifunctional agents for Alzheimer’s disease (AD)

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therapy, and their potential was evaluated through various biological experiments. Among these derivatives, compound 1b displayed good selective inhibitory activity against AChE (vs BuChE, IC50 = 0.324 µM, SI > 123) and MAO-B (vs MAO-A, IC50 = 1.427 µM, SI > 35). Molecular docking study showed that the pyrimidinylthiourea

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moiety of 1b could bind to the catalytic active site (CAS) of AChE, and the propargylamine moiety interacted directly with the flavin adenine dinucleotide (FAD)

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of MAO-B. Moreover, 1b demonstrated mild antioxidant ability, good copper chelating property, effective inhibitory activity against Cu2+-induced Aβ1−42 aggregation, moderate neuroprotection, low cytotoxicity, and appropriate blood−brain barrier

(BBB)

permeability

in

vitro

and

was

capable

of

ameliorating

scopolamine-induced cognitive impairment in mice. These results indicated that 1b

disease.

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has the potential to be a multifunctional candidate for the treatment of Alzheimer’s

Keywords: Multifunctional agents, AChE inhibitors, MAO-B inhibitors, Metal

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chelating agents, Alzheimer’s disease.

ACCEPTED MANUSCRIPT 1. Introduction Alzheimer’s disease (AD) is a progressive and chronic neurodegenerative disorder characterized by memory loss and cognitive impairments [1,2]. The number of dementia patients has reached over 46 million by 2015 and is expected to triple by

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2050 [3]. Due to its complex etiology, the pathogenesis has not been fully elucidated, and there have been various pathogenesis hypotheses for AD, such as cholinergic hypothesis [4], amyloid cascade hypothesis [5,6], oxidative stress hypothesis [7], and metal dishomeostasis hypothesis [8,9].

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Among these hypotheses, the cholinergic deficit hypothesis has been generally accepted by most researchers. This theory suggests that AD patients’ learning-memory

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and cognitive ability dysfunction is closely related to the low acetylcholine (ACh) levels in brain [10,11]. Thus, strengthening the central cholinergic activity and increasing the level of ACh in the brain, for example, by inhibiting the activity of acetylcholinesterase (AChE) have been considered to be effective approaches for the treatment of AD [12,13]. Currently, the clinical first-line drugs for the treatment of

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AD are mainly AChE inhibitors such as donepezil, rivastigmine, galantamine, and huperzine A (Hup A, approved by CFDA) [14,15]. However, these drugs only improve the memory and cognitive abilities of AD patients but fail to achieve a

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radical cure [16,17].

Many studies have found that monoamine oxidase (MAO) also plays a very important role in the pathogenesis of AD because the increase of MAO in the brain

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may result in a cascade of biochemical events leading to neuronal dysfunction [18,19]. Monoamine oxidases (MAOs) are flavin adenine dinucleotide (FAD)-containing enzymes that are responsible for the oxidative deamination of endogenous and exogenous monoamine substances. MAOs have two functional isozymic forms, namely, MAO-A and MAO-B [20]. MAO-A inhibitors are used as clinical antidepressants and anti-anxiety agents, whereas MAO-B inhibitors are applied to treat neurodegenerative disorders such as AD and Parkinson’s diseases (PD) [21,22]. According to previous studies [23,24], the activities of MAO-B in the brain and blood platelets of AD patients were increased, while high expression levels of MAO-B

ACCEPTED MANUSCRIPT could cause the high level of free radicals that played a key role in the pathogenesis of AD. MAO-B inhibitors can reduce the oxidative stress response and protect the nerve cells from oxidative damage and neurotoxicity, and therefore, MAO-B has been another important target for the treatment of AD [25,26]. Among the MAO inhibitors,

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selegiline, an irreversible and selective MAO-B inhibitor, has been reported as a potential anti-AD agent due to its neuroprotective property in cellular and animal models of AD [27].

In addition, recent studies have revealed that the high levels and dysregulation of

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biometal ions were closely implicated in the pathogenesis of AD [8,9,28]. Metal ions such as Cu2+, Zn2+ and Fe2+ have been shown to facilitate Aβ aggregation, leading to

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the generation of toxic Aβ oligomers [29]. In particular, redox-active Cu (I/II) and Fe (II/III) are implicated in the generation of reactive oxygen species (ROS) leading to an increased oxidative stress [30-33]. Biometal chelators, especially Cu2+ chelators, not only reduce the metal-induced Aβ aggregation but also reduce the level of ROS produced by the redox metal and metal-Aβ complex [33,34]. Therefore, biometal

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chelators have been considered as a potential therapeutic approach for AD. Furthermore, neurotoxic ROS and oxidative damage of neuronal cells are also associated with AD, so that compounds with antioxidant activities are beneficial for

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the treatment of AD [35,36].

Taking the complex pathogenesis of AD and the impotency of single-target drugs into consideration, the development of multi-target-directed ligands (MTDLs) has

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been proposed to be an effective approach for the treatment of AD [37,38]. Considering that ChEs and MAOs are important targets for the treatment of AD, some researchers have devoted their efforts to finding multifunctional agents that target both ChEs and MAOs. For example, Ladostigil (Fig. 1), generated by joining the carbamate moiety of rivastigmine and indolamine moiety of rasagiline, shows bifunctional inhibitory activities of MAOs and ChEs in a single molecule, and presently, it is in a Phase III clinical trial for the treatment of mild cognitive impairment (MCI). This indicates that the combination of ChEs inhibitor and MAOs inhibitor is a promising therapeutic method for AD [39-41].

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Fig. 1. Chemical structures of ladostigil, rivastigmine and rasagiline

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Inspired by the MTDLs strategy, we designed and synthesized a series of novel pyrimidinylthiourea derivatives as multifunctional agents for the potential treatment of AD in our previous work. These derivatives exhibited potent inhibition and good selectivity toward AChE, specific metal-chelating ability, mild antioxidant effects,

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low cytotoxicity, and good blood–brain barrier (BBB) permeability and could improve memory and cognitive function of scopolamine-induced cognitive

pyrimidinylthiourea

moiety

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impairment mice [42]. In this work, we combined the imidazole-substituted (AChE

inhibitor’s

pharmacophore)

and

the

propargylamine moiety (MAO-B inhibitor’s pharmacophore) of selegiline [27] or pargyline [43] into a single molecule (Fig. 2). These derivatives showed good inhibition for both AChE and MAO-B, as well as the abilities of metal-chelating,

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modulating metal induced Aβ aggregation, free radical scavenging, mild antioxidant

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properties, neuroprotection and low cytotoxicity.

Fig. 2. Design strategy for the new series of propargylamine-modified pyrimidinylthiourea derivatives. 2. Results and discussion 2.1. Design and synthesis To study the SAR of new compounds and find the optimal compound, structural

ACCEPTED MANUSCRIPT modifications were performed. First, “R” at the N-atom of propargylamine was modified with hydrogen, methyl, ethyl, propyl, butyl, acetyl, benzyl, cyanomethyl and propargyl to study the effect of different sizes of substitution on multiple functions (compounds 1a-i). Next, to obtain the optimal linker between imidazole ring and

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propargylamine, the length of carbon spacer (“n”) was changed from one to three or a ring (compounds 2a-d, 3a-b, and 1j). The synthesis of propargylamine-modified pyrimidinylthiourea derivatives (1-3) was carried out following the sequence of reactions depicted in Scheme 1. The nucleophilic substitution reaction of imidazole

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derivatives (4a-c) and 4-amino-6-chloropyrimidine in the presence of inorganic base (K2CO3 or Cs2CO3) provided intermediates 5a-c, followed by the condensation

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reaction with ethylisothiocyanate in the presence of NaH affording the key intermediates 6a-c. The target compounds 1a-b and 1j were obtained by reductive amination of intermediate 6a with diverse propargylamines in the presence of NaBH3(CN). Next, the nucleophilic substitution reaction of 1a with haloalkane or acetylchloride in the presence of Et3N provided compounds 1c-i. To obtain

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compounds 2a-d and 3a-b, the intermediates 7b-c were synthesized by the bromination reaction of 6b-c with PBr3. Then, the nucleophilic substitution of 7b-c with propargylamine or N-methyipropargaylamine afforded the target compounds

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2a-b and 3a-b. Similarly, the nucleophilic substitution reaction of 2a with haloalkane in the presence of Et3N furnished the target compounds 2c-d. The details of the synthetic procedures and structural characterizations of target

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compounds (1H NMR,

13

C NMR, and HRMS spectroscopy) and intermediates (1H

NMR) are described in the experimental section. The purities of all target compounds were determined by HPLC.

ACCEPTED MANUSCRIPT R1

R1

N

N N

4a-c

H N

b

N

H N S

N

5a-c

6a: R1 = CHO 6b: R1 = CH2CH2OH 6c: R1 = CH2CH2CH2OH

N

CHO H N

N N

S

R2 c

N

H N

N

H N S

N

CHO

1a: R2 = H 1b: R2 = CH3 1c: R2 = CH2CH3 1d: R2 = CH2CH2CH3 1e: R2 = CH2CH2CH2CH3 1f: R2 = C(O)CH3 1g: R2 = CH2Ph 1h: R2 = CH2CN 1i: R2 = CH2CCH

N N

N

N

c

N

H N

N N

H N

N

N

N

N

1j

OH n

S

H N

S

6a

N

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H N

H N

N

1a-i

d

S

N

N

6a

H N

N

6a-c

4a: R1 = CHO 5a: R1 = CHO 4b: R1 = CH2CH2OH 5b: R1 = CH2CH2OH 4c: R1 = CH2CH2CH2OH 5c: R1 = CH2CH2CH2OH

H N

N

N

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H2N

a

N

SC

HN

R1

Br n

e

N

N

H N

S

6b-c

N

N

N

N n R3

H N

N

7b-c

7b: n = 2 7c: n = 3

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6b: n = 2 6c: n = 3

H N

f

d

H N S

N N

N

N

2a-d, 3a-b 2a: n = 2, R3 = H 2b: n = 2, R3 = CH3 2c: n = 2, R3 = CH2CH3 2d: n = 2, R3 = CH2CN 3a: n = 3, R3 = H 3b: n = 3, R3 = CH3

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Scheme 1. Synthetic route to compounds 1a-j, 2a-d and 3a-b. Reagents and conditions: (a) (i) for 5a: 4-amino-6-chloropyrimidine, K2CO3, DMF, 100 °C, 12 h; (ii) for 5b-c: 4-amino-6-chloropyrimidine, Cs2CO3, DMF, 140 °C, 12 h; (b) CH3CH2NCS, DMF,

rt,

0.5

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NaH,

h;

(c) 2-Propynylamine,

N-methylpropargylamine or

1-(prop-2-yn-1-yl)piperazine, NaBH3(CN), MeOH:DCE = 1:1 (v:v), AcOH, rt, 12 h; (d) (i) for 1c-e and 1g-i: RX, acetonitrile, Et3N, reflux, 12 h; (ii) for 1f: acetyl chloride, DCM, Et3N, rt; (e) PBr3, DCM, rt, overnight; (f) 2-Propynylamine or N-methylpropargylamine, K2CO3, rt, overnight. 2.2. Biological activity 2.2.1. In vitro inhibition studies of AChE and BuChE To evaluate the therapeutic potential of these propargylamine-modified pyrimidinylthiourea derivatives for AD, compounds 1a−j, 2a−d, and 3a−b were

ACCEPTED MANUSCRIPT assayed against rat ChE (rChE) by the modified method of Ellman et al. [44], using huperzine A (Hup A) as a reference compound. As shown in Table 1, all of our target compounds exhibited desirable inhibitory activity to rAChE and poor inhibitory activity against rBuChE (SI> 30), indicating that these derivatives were selective

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AChE inhibitors. Examination of the IC50 values of compounds 1a-i shows that unsubstituted compound 1a (IC50 = 0.354 µM for AChE) and methyl substituted compound 1b (IC50 = 0.324 µM for AChE) exhibited higher activity than compounds 1c-i (IC50 > 0.4 µM for AChE) with larger substitutions. This suggested that the size

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of the substitution at the N-atom of propargylamine has a moderate influence on the AChE inhibitory activity, and the appropriate substituents at the N-atom of

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propargylamine appeared to be methyl and hydrogen. Then, the influence of the linker length to AChE inhibitory activity was also studied; however, compounds 1a-b, 2a-b and 3a-b exhibited similar inhibitory activities for AChE. The inhibitory activity result of compound 1j shows that the use of the piperazine ring as the linker led to a decrease in AChE inhibitory activity.

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Table 1.

Structures, rAChE and rBuChE inhibitory activities, and selectivity index for rAChE

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of the target compounds.

Compd

n

IC50 (µM)a for

IC50 (µM)a for rBuChE

rAChE

(Inhibition at 40 µM)

R

SIb

1

H

0.354 ± 0.057

>40 (15.24%)

>113

1

CH3

0.324 ± 0.022

>40 (12.59%)

>123

1c

1

CH2CH3

0.482 ± 0.019

>40 (10.49%)

>83

1d

1

(CH2)2CH3

0.485 ± 0.025

>40 (7.75%)

>83

1e

1

(CH2)3CH3

0.675 ± 0.072

>40 (13.42%)

>59

1f

1

C(O)CH3

0.515 ± 0.070

>40 (5.15%)

>78

1a 1b

1

CH2Ph

0.585 ± 0.012

>40 (9.28%)

>68

1h

1

CH2CN

0.707 ± 0.044

>40 (5.48%)

>57

1i

1

CH2CCH

0.718 ± 0.034

>40 (4.97%)

>56

2a

2

H

0.228 ± 0.027

>40 (8.73%)

>175

2b

2

CH3

0.392 ± 0.037

>40 (8.91%)

>102

2c

2

CH2CH3

1.337 ± 0.184

>40 (2.44%)

>30

2d

2

CH2CN

0.597 ± 0.011

>40 (3.02%)

>67

3a

3

H

0.381 ± 0.052

>40 (12.74%)

>105

3b

3

CH3

0.459 ± 0.052

>40 (6.70%)

>87

1j

0.482 ± 0.029

>40 (7.30%)

>83

Hup A

0.098 ± 0.016

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1g

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36.4 ±1.838

>371

a

Results are expressed as the mean ± SD of at least three independent experiments.

b

Selectivity index for AChE is defined as IC50(rBuChE)/IC50(rAChE).

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2.2.2. In vitro inhibition studies of MAOs

To confirm the multipotent biological functions of these new compounds, inhibitory activities against the recombinant human MAOs (hMAO-A and hMAO-B) were determined using clorgyline and pargyline as the reference compounds,

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respectively. The corresponding IC50 values or the percentage of inhibition at 50 µM are shown in Table 2. The data showed that only a fraction of the tested compounds

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could effectively inhibit MAO-A or MAO-B. Among the synthesized compounds, 1b was the most potent selective inhibitor against MAO-B (IC50 = 1.472 µM, SI > 35), and 1a exhibited the highest potency for both MAO-A and MAO-B (IC50 = 4.327 µM and 5.236 µM, respectively). The other compounds, except 1a-b, 2b and 3b, showed weak activities with IC50 values over 50 µM. This result suggested that the enlargement of the substitution at the N-atom of propargylamine dramatically decrease the inhibitory activity for MAOs. For unsubstituted compounds (1a, 2a and 3a), once the linker length changed from 1 to 3 carbon atoms, the inhibitory activities for MAO-A and MAO-B decreased sharply (1a: IC50 = 4.327 µM for MAO-A, IC50 =

ACCEPTED MANUSCRIPT 5.236 µM for MAO-B; 2a and 3a: IC50 > 50 mM for both MAO-A and MAO-B); for N-methyl substituted compounds (1b, 2b and 3b), the linker length affected the selectivity toward MAO-A and MAO-B: compound 2b with a 2 carbon atoms-long linker exhibited inhibitory activities for both MAO-A and MAO-B, whereas 1b and

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3b, with 1 and 3 carbon atoms-long linkers, respectively, were good selective inhibitors against MAO-B. This meant that the selectivity of the compounds towards MAO-A and MAO-B is closely related to the length of the linker. Table 2.

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Structures, hMAO-A and hMAO-B inhibitory activities, and selectivity index for

n

R

1

H

1b

1

CH3

1c

1

CH2CH3

1d

1

(CH2)2CH3

1e

1

1f

IC50 (µM)a for

hMAO-A

hMAO-B

(Inhibition at 50 µM)

(Inhibition at 50 µM)

SIb

ORACc

4.327 ± 0.299

5.236 ± 0.527

0.826

1.175 ± 0.061

>50 (20.22%)

1.427 ± 0.272

>35

1.126 ± 0.015

>50 (6.71%)

>50 (35.12%)

1.383 ± 0.061

>50 (1.76%)

>50 (24.31%)

0.448 ± 0.222

(CH2)3CH3

>50 (2.43%)

>50 (20.11%)

0.238 ± 0.013

1

C(O)CH3

>50 (4.83%)

>50 (1.17%)

1.167 ± 0.035

1

CH2Ph

>50 (17.82%)

>50 (18.44%)

n.t.

1

CH2CN

>50 (2.01%)

>50 (15.35%)

0.494 ± 0.031

1

CH2CCH

>50 (15.47%)

>50 (42.11%)

0.585 ± 0.070

2a

2

H

>50 (54.24%)

>50 (19.23%)

1.272 ± 0.053

2b

2

CH3

9.003 ± 0.982

12.435 ± 1.082

2c

2

CH2CH3

>50 (13.03%)

>50 (49.4%)

n.t.

2d

2

CH2CN

>50 (13.92%)

>50 (24.94%)

0.556 ± 0.029

3a

3

H

>50 (31.39%)

>50 (36.59%)

0.186 ± 0.004

1h 1i

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1g

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1a

IC50 (µM)a for

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Compd

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hMAO-B of the target compounds.

0.724

1.243 ± 0.016

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3.229 ± 0.557

1j

>50 (9.22%)

>50 (13.29%)

1.028 ± 0.061

Clorgyline

12.850 ± 0.495（nM）

n.t.d

n.t.

Pargyline

n.t.

0.188 ± 0.061

a

CH3

>15

0.447 ± 0.169

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3

3b

n.t.

Results are expressed as the mean ± SD of at least two experiments. bSelectivity

index for MAO-B is defined as IC50(MAO-A)/IC50(MAO-B). cThe mean ± SD of

of tested compounds. dNo test.

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2.2.3. Free oxygen radical scavenging ability

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three independent experiments. Data are expressed as µmol of Trolox equivalent/µmol

Given that reactive radical species have been identified to be closely related to AD [45], compounds that can decrease reactive radical species may exert potential therapeutic effects. The oxygen radical absorbance capacity assay (ORAC-FL) was performed to evaluate the antioxidant activity of these target compounds. This assay fluorescein

as

the

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used

fluorescent

probe,

AAPH

(2,2’-azobis-(amidinopropane)dihydrochloride) as the peroxyl radical inducer, and the water-soluble analogue of vitamin E, Trolox, as the standard [46,47]. ORAC values

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were expressed as Trolox equivalents calculated from the antioxidant of tested compounds vs Trolox (the ORAC value of Trolox = 1.00). The results (Table 2)

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indicated that half of the tested compounds exhibited mild antioxidant ability with ORAC values of 1.028−1.383. The modification of the “R” substituent at the N-position and changes in the length of the linker obviously affected the antioxidant activity (1c vs 1d-e; 1a and 2a vs 3a): unsubstituted and small-size substituent compounds with 1−2 carbon atoms linker length exhibited relatively good antioxidant activity (ORAC values > 1), whereas the rest of the tested compounds displayed weak antioxidant activity (ORAC values < 1). Compound 1c displayed the best antioxidant activity, with an ORAC value of 1.383. 2.2.4. Metal chelating effect

ACCEPTED MANUSCRIPT Based on above biological evaluation results, compound 1b was the most promising compound with excellent multiple functions (IC50 = 0.324 µM for AChE; IC50 = 1.472 µM for MAO-B; ORAC value of 1.126 for antioxidant activity). Therefore, compound 1b was chosen for further biological evaluation.

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The metal chelating activity of compound 1b with copper, zinc, and iron was measured by UV−vis spectroscopy assay. In our previous work [42], we found that thiourea fragment played the key role in the generation of the metal chelating action, and the pyrimidinylthiourea derivatives could selectively chelate Cu2+. As expected,

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when CuCl2 was added to the solution of 1b, the maximum absorption decreased dramatically at 290 nm (Fig. 3), demonstrating the formation of 1b−Cu2+ complexes,

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while only vague optical shift and maximum absorption fluctuation were observed when FeSO4, FeCl3, and ZnCl2 were added, indicating that compound 1b has better

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selectivity towards Cu2+.

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Fig. 3. UV spectra of compound 1b (40 µM) alone and in the presence of CuCl2 (20 µM), FeSO4 (20 µM), FeCl3 (20 µM), or ZnCl2 (20 µM) in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). 2.2.5. Inhibition of ROS produced by copper redox cycling Many researchers have found that redox-active metal ions were closely involved in the high oxidative stress caused by ROS [31], which is a proposed pathogenesis of AD. To evaluate the ability of compound 1b to inhibit the ROS produced by Cu(II)-related redox, the model of the Cu-ascorbate redox system described by Faller and

co-workers

was

used

(Fig.

4A)

[48],

using

the

metal

chelator

ACCEPTED MANUSCRIPT ethylenediaminetetraacetic acid (EDTA) as the positive control. Hydroxyl radicals (OH·), produced by copper redox cycling in the presence of ascorbate, could oxidize coumarin-3-carboxylic acid (CCA) to fluorescent 7-hydroxy CCA so that the fluorescence values could indicate the amount of OH·. As shown in Fig. 4B, the

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fluorescence value of the copper-ascorbate system increased steadily with time and reached a plateau at approximately 800 s. However, the value changed little when compound 1b was coincubated with the Cu-ascorbate system and was the same as the value of the sample without Cu(II). The result showed that compound 1b could halt

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copper redox cycling.

Fig. 4. (A) Oxygen reduction by copper redox cycling in the presence of ascorbate. (B) intensity

of

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Fluorescence

the

copper-ascorbate-1b,

copper-ascorbate-EDTA,

copper-ascorbate, and ascorbate alone systems. CCA (50 µM) and ascorbate (150 µM)

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were incubated in each system. [Cu2+] = 5 µM, [1b] = [EDTA] = 15 µM. PBS buffer (20 mM KH2PO4, 150 mM NaCl); N = 3; pH = 7.4; 37 °C. 2.2.6. Dot blot assay and TEM assay A dot blot assay was performed to evaluate the modulation of metal-induced Aβ

aggregation by compound 1b, and a transmission electron microscopy (TEM) assay was then performed to examine the morphological changes of the Aβ species. In the dot blot assay, the samples were probed by the A11 antibody to determine the oligomeric Aβ, and total Aβ was detected by 6E10 as the loading control. A representative scan graph and a statistical histogram are shown in Fig. 5A; the gray

ACCEPTED MANUSCRIPT density of the spots is proportional to the level of the Aβ oligomerization. The results showed that Aβ aggregated spontaneously, and compound 1b showed no effect on the Aβ self-aggregation after 24 h incubation. Epigallocatechin gallate (EGCG) was tested as the positive control. A thioflavine T (ThT) fluorescence assay was performed

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to verify this result (Fig. S1, Supporting Information). The dot blot assay showed that Cu2+ could accelerate Aβ aggregation and that this process was significantly inhibited by compound 1b.

The TEM experiment showed that the aggregation of Aβ in the presence of Cu2+

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produced more fibrils than self-induced Aβ aggregation (Fig. 5B, images 2 and 3). As expected, fewer Aβ fibrils were detected when compound 1b and clioquinol (CQ)

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were incubated with the sample after incubation for 24 h at 37 °C (Fig. 5B, images 4

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and 5), indicating that 1b can efficiently inhibit Cu2+-induced Aβ aggregation.

Fig. 5. Inhibition of compound on Cu2+-induced Aβ1−42 aggregation. (A) Scan and bar graph of the dot blot assay. Aggregated Aβ was detected by A11 antibody, and total Aβ was probed by 6E10 antibody. Data are shown as the mean ± SEM, ###p < 0.001 vs Aβ fresh, *p < 0.05, **p < 0.01 vs Aβ alone, $p < 0.05, $$p < 0.01 vs Aβ + Cu2+, N = 4. (B) TEM image analysis of copper induced Aβ1−42 aggregation studies ([Aβ1−42] = [Cu2+] = 25 µM, [compd] = 50 µM; HEPES (20 µM) and NaCl (150 µM); pH 6.6; 37 °C). (1) fresh Aβ1−42, (2) Aβ1−42 + Cu2+, (3) Aβ1−42 alone, (4) Aβ1−42 + Cu2+ + 1b, (5) Aβ1−42 + Cu2+ + CQ.

ACCEPTED MANUSCRIPT 2.2.7. MTT Assay To investigate the safety of the propargylamine-modified 4-aminoalkyl imidazole substituted pyrimidinylthiourea derivatives, the cytotoxicity of compound 1b on rat primary

cortical

neuron

(PCN)

was

studied

using

a

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and donepezil (Don) tested as the reference. The result is depicted in Fig. 6A. Similar to Don, compound 1b exhibited no cytotoxicity at concentrations of 10 µM and 30 µM after incubation for 24 h. When the concentration increased to a relatively high level

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of 100 µM, 1b showed a significant but mild neurotoxicity (*p < 0.05 vs Ctrl) compared to Don (***p < 0.001 vs Ctrl). Therefore, the MTT assay showed that

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compound 1b was weakly cytotoxic.

Neurotoxicity, neuronal damage and neuronal cell apoptosis caused by the toxic Aβ oligomer are important causes of AD. To determine whether compound 1b can protect neuronal cells from toxic Aβ species, cell viability experiments were conducted using PCNs. EGCG (inhibitor of Aβ self-aggregation) and CQ (inhibitor of

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Cu2+-induced Aβ aggregation) were tested as positive controls. The results are shown in Fig. 6B. Incubation of PCNs with Aβ in the presence of Cu2+ caused higher cytotoxicity than the Aβ alone group (*p < 0.05). Compared to the Aβ + Cu2+ group, compound 1b, like CQ, could protect the PCNs from Cu2+-induced Aβ neurotoxicity,

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leading to a significant increase in cell viability ($$p < 0.01). Moreover, compound 1b could not interact with Aβ directly to restrain the cytotoxicity produced by the Aβ-self

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aggregates. These results were highly consistent with the results of the dot blot assay.

ACCEPTED MANUSCRIPT Fig. 6. (A) Cell viability of primary cortical neurons exposed to compound 1b and Don at diff erent concentrations (range 10−100 µM) for 24 h. Vehicle-treated cells were used as controls. The results are expressed as the percentage of viable cells observed after treatment with compound vs vehicle-treated cells (100%) and are ***

p < 0.001 vs Ctrl, N = 3; (B) Protection of

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shown as the mean ± SEM; *p < 0.05,

compound 1b from Cu2+-induced Aβ1-42 neurotoxicity. Data are shown as the mean ± SEM; N = 3. Experimental conditions: [Aβ1−42] = [Cu2+] =1 µM, [compd] = 2 µM. ###

p < 0.001 vs Ctrl, *p < 0.05 vs Aβ alone, $$p < 0.01 vs Aβ + Cu2+.

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2.2.8. In vitro blood−brain barrier permeation assay

To evaluate the target compounds’ BBB penetrability, we carried out the parallel

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artificial membrane permeation assay (PAMPA) [49]. First, to validate the assay, the values of permeability (Pe) of 13 commercial drugs were determined and compared to the reported values. A plot of the experimental data versus reference values gave a strong linear correlation, Pe (exp.) = 0.947Pe (bibl.) – 0.8152 (R2 = 0.9651, Fig. S2, Supporting Information). Based on this equation and the limit established by Di et al.

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for blood−brain barrier permeation, we concluded that compounds with Pe values greater than 3.08 × 10−6 cm s−1 could cross the blood−brain barrier by passive permeation (CNS+). Pe values from 1.13×10−6 cm s−1 to 3.08 × 10−6 cm s−1 were

EP

classified as “CNS±”, uncertain BBB permeation. The results of PAMPA-BBB assay are presented in Table 3. It can be observed that compounds 1b, 2a-b and 3a-b could cross the BBB, whereas compound 1a exhibited uncertain BBB permeation (CNS±).

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It can also be seen that the compounds’ BBB permeation ability is related to the length of the linker (3b > 2b > 1b). Table 3.

Permeability results (Pe×10−6 cm s−1) from the PAMPA-BBB assay for compounds 1a-b, 2a-b and 3a-b and their prediction of BBB penetration. Compd

Pe (10−6 cm s−1)a

predictionb

1a

2.78 ± 0.12

CNS±

1b

3.16 ± 0.10

CNS+

ACCEPTED MANUSCRIPT 2a

3.24 ± 0.08

CNS+

2b

3.33 ± 0.12

CNS+

3a

3.26 ± 0.21

CNS+

3b

3.98 ± 0.09

CNS+

Values are expressed as the mean ± SD of at least three independent experiments.

b

Compounds with Pe > 3.08 × 10−6 cm s−1 could cross the BBB by passive diffusion

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a

(CNS+). Compounds with Pe < 1.13 × 10−6 cm s−1 could not cross the BBB (CNS−), and compounds with 1.13 × 10−6 cm s−1 < Pe < 3.08 × 10−6 cm s−1 show uncertain

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BBB permeation (CNS±).

2.2.9. Improvement of cognition in scopolamine-induced dementia mice by 1b.

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To analyze the in vivo effect of compound 1b on cognitive impairment, scopolamine-induced cognitive performance deficits in the channel water maze were employed, with huperzine A (Hup A) as a positive control. As shown in Fig. 7, mice treated with scopolamine (4.5 mg/kg, i.p.) required longer time to find the platform (escape latency) and committed more errors (entering no-exit paths) than those of the

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control group (###p < 0.001) in the water maze task. Compound 1b·HCl, administered to mice orally at a dose of 30 mg/kg, could ameliorate scopolamine-induced learning and memory deficits, with the mice in this group showing shorter escape latency and

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less frequent errors compare to the scopolamine group (*p < 0.05). Therefore, compound 1b·HCl has the potential to be a lead anti-AD agent due to its symptomatic

AC C

improvement effect in the AD mice model.

Fig. 7. Effect of compound 1b·HCl (30 mg/kg) on scopolamine-induced cognitive deficit mice, with Hup A (0.1 mg/kg) as positive control: (A) Escape latency of mice

ACCEPTED MANUSCRIPT in water maze; (B) Number of errors of mice in water maze. Data are shown as the mean ± SEM: ###p < 0.001 vs Ctrl, *p < 0.05, **p < 0.01, ***p < 0.001 vs Scop, N = 16. Ctrl: control; Scop: scopolamine; HupA: huperzine A. 2.3. Molecular docking studies of AChE and MAO-B

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To confirm the rationality of compound design and explore the interaction mode of target compounds with enzymes, molecular docking simulations of 1b with mice AChE (mAChE) and human MAO-B (hMAO-B) were performed using Glide in Schrödinger software. The X-ray crystallographic structure of mAChE complexed tacrine

(PDB

code

5EIE),

and

human

MAO-B

complexed

with

SC

with

7-(3-chlorobenzyloxy)-4-formylcoumarin (PDB code 2V60) were obtained from the

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Protein Data Bank. As shown in Fig. 8A, compound 1b interacts with the CAS of AChE via π-π interactions and hydrogen bond interactions. The two strong hydrogen bonds between the thiourea-NH (1.7 Å) and carbonyl of His 447 (2.2 Å) may play an important role in the positioning and the stabilization of the ligand inside the CAS. Moreover, its pyrimidine ring could interact with Trp86 via face-to-face π-π stacking

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interaction with the distance of 4.1 Å. The results also validated the hypothesis that the thiourea and pyrimidine fragments were key groups for maintaining the AChE activity [42].

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Examination of Fig. 8B shows that the propargylamine group of 1b has a favorable fit into the substrate cavity of the enzyme, oriented to the flavin adenine dinucleotide (FAD) cofactor in close proximity (4.64 Å), and is properly adopted

AC C

between Tyr398 (3.32 Å) and Tyr435 (3.29 Å), forming face-to-face cation-π stacking interactions in a “sandwich” form. Additionally, the face-to-edge π-π stacking interaction (4.9 Å) between the pyrimidine ring and Trp326 and the hydrogen bond (2.0 Å) between thiourea-NH and carbonyl of Ile199 could stabilize the ligand in the catalysis gorge of the enzyme. Moreover, the ethyl group of thiourea may be embedded into the hydrophobic pocket in the entrance cavity formed by Pro104, Phe103, Pro102, Ile199, Ile316, Leu167 and Phe168.

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ACCEPTED MANUSCRIPT

Fig. 8. (A) 3D docking model of compound 1b with mAChE. (B) 3D docking model

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of compound 1b with hMAO-B. Compound 1b is illustrated using blue sticks, and the FAD cofactor is depicted using yellow sticks. π-π stacking interactions are highlighted by green lines, and hydrogen bonds are shown with purple dash lines. 3. Conclusions

Multi-target-directed ligands (MTDLs), aiming to simultaneously target multiple

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pathological processes involved in the neurodegenerative cascade, have been developed as effective agents for the treatment of Alzheimer’s disease. Our previous studies obtained the pyrimidinylthiourea derivatives as MTDLs with ChE inhibitory activity, specific metal-chelating ability and mild antioxidant effect. In the present

EP

study, by introducing the MAO-B inhibitor’s pharmacophore propargylamine into the pyrimidinylthiourea scaffold, a series of novel propargylamine-modified 4-aminoalkyl substituted

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imidazole

pyrimidinylthiourea

derivatives

have

been

designed,

synthesized, and evaluated as potential multifunctional anti-AD agents. The activities of all synthesized compounds were evaluated against rAChE, rBuChE, hMAO-A(B), and antioxidant ability. The SAR studies revealed that the size of the substitution at the N-atom of propargylamine strongly influences the AChE and MAOs inhibition and the antioxidant activity. Among all derivatives, compound 1b displayed good selective inhibitory activities against AChE (IC50 = 0.324 µM, SI > 123) and MAO-B (IC50 = 1.427 µM, SI > 35) and demonstrated mild antioxidant ability (ORAC = 1.126), good copper chelating property, effective inhibitory activity against

ACCEPTED MANUSCRIPT Cu2+-induced Aβ1−42 aggregation, low cytotoxicity and moderate neuroprotection against Cu2+-induced Aβ1−42 neurotoxicity in primary cultured cortical neurons and appropriate blood−brain barrier (BBB) permeability in vitro. The molecular docking simulations of 1b with mAChE and hMAO-B also confirmed the rationality of our

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design strategy. Furthermore, compound 1b·HCl could improve the memory and cognitive function of Scop-induced dementia mice. Taken together, these results showed that the new compound 1b can be considered a potential multifunctional agent for AD therapy.

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4. Experimental section 4.1. Chemistry

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4.1.1. General methods

All common reagents and solvents were obtained from commercial suppliers and used without purification. Reaction progress was monitored using analytical thin layer chromatography (TLC), HSGF 254 (150−200 µm thickness; Yantai Huiyou Co., China), and the spots were detected under UV light (254 nm). Melting points were

1

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measured in capillary tubes on a SGWX−4 melting point apparatus without correction. H NMR and 13C NMR spectra were recorded on a Bruker AMX 400 and AMX 500

spectrometer in DMSO-d6, CD3COCD3 or CD3OD with TMS as internal standard.

EP

Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). High-resolution mass spectra

AC C

(HRMS) were obtained by electric ionization (EI) and electrospray ionization (ESI) using a Waters GCT Premie and Waters LCT. All target compounds were purified to ≥ 95% purity as determined by an Agilent 1100 with a quaternary pump and diode array detector (DAD). The eluent was CH3OH:H2O = 80:20 (v:v), flow rate was 0.5 mL/min, and the relative purity of each compound calculated at 254 nm. 4.1.2. General synthetic procedure for 5a-c A

mixture

of

imidazole

derivatives

(4a-c,

10

mmol),

4-amino-6-chloropyrimidine (1.30 g, 10 mmol), and K2CO3 (2.07 g, 15 mmol, for 5a) or Cs2CO3 (3.91 g, 12 mmol, for 5b-c) was stirred in DMF (20 mL) at 100 °C (for 5a)

ACCEPTED MANUSCRIPT or at 140 °C (for 5b-c) for 12 h. Water (100 mL) was then added. The mixture was extracted with EtOAc (3 × 50 mL). The organic layer was separated and dried over Na2SO4. Removal of the solvents produced a residue which was purified using column chromatography and eluted with a mixture of MeOH:CH2Cl2 (1:30, v:v) to

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afford 5a-c. 4.1.2.1. 1-(6-Aminopyrimidin-4-yl)-1H-imidazole-4-carbaldehyde (5a) [42]. White solid, 46% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.70 (s, 1H), 8.64 (s, 1H), 8.37 (s, 1H), 7.35 (s, 2H), 6.72 (s, 1H).

SC

4.1.2.2. 2-(1-(6-Aminopyrimidin-4-yl)-1H-imidazol-4-yl)ethan-1-ol (5b). White solid, 58% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.29 (s, 1H), 7.54 (s,

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1H), 7.16 (s, 2H), 6.50 (s, 1H), 4.64 (br, 1H), 3.70–3.60 (m, 2H), 2.66 (t, J = 6.9 Hz, 2H).

4.1.2.3. 3-(1-(6-Aminopyrimidin-4-yl)-1H-imidazol-4-yl)propan-1-ol (5c). White solid, 41% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 1.1 Hz, 1H), 8.29 (s, 1H), 7.50 (s, 1H), 7.13 (s, 2H), 6.50 (d, J = 0.7 Hz, 1H), 4.47 (t, J = 5.2 Hz, 1H), 3.50

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– 3.38 (m, 2H), 2.59–2.51 (m, 2H), 1.81–1.69 (m, 2H). 4.1.3. General synthetic procedure for 6a-c

NaH (60% dispersion in mineral oil, 96 mg, 2.4 mmol) was added to a solution

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of corresponding intermediates 5a-c (2 mmol) in DMF (10 mL). The solution was stirred for 5 min, and then, ethyl isothiocyanate (174 mg, 2 mmol) was added. The reaction mixture was stirred at room temperature for 0.5 h. Water was added, and the

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mixture was extracted with EtOAc (3 × 60 mL). The organic layer was separated and dried over Na2SO4. Removal of the solvents produced a residue, which was purified using column chromatograph and eluted with a mixture of MeOH:CH2Cl2 (1:40, v:v) to afford corresponding intermediates 6a-c. 4.1.3.1. 1-Ethyl-3-(6-(4-formyl-1H-imidazol-1-yl)pyrimidin-4-yl)thiourea (6a) [42]. White solid, 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.13 (t, J = 5.1 Hz, 1H), 11.10 (s, 1H), 9.88 (s, 1H), 8.83 (s, 1H), 8.61 (s, 2H), 7.42 (s, 1H), 3.72–3.57 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H). 4.1.3.2.

1-Ethyl-3-(6-(4-(2-hydroxyethyl)-1H-imidazol-1-yl)pyrimidin-4-yl)

ACCEPTED MANUSCRIPT thiourea (6b). White solid, 41% yield. 1H NMR (400 MHz, CD3OD) δ 8.69 (d, J = 0.9 Hz, 1H), 8.52 (d, J = 1.2 Hz, 1H), 7.64 (s, 1H), 6.98 (d, J = 0.9 Hz, 1H), 3.84 (t, J = 6.7 Hz, 2H), 3.74 (q, J = 7.3 Hz, 2H), 2.82 (t, J = 6.7 Hz, 2H), 1.31 (t, J = 7.3 Hz, 3H). 1-ethyl-3-(6-(4-(3-hydroxypropyl)-1H-imidazol-1-yl)pyrimidin-4-yl)

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4.1.3.3.

thiourea (6c). White solid, 35% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.19 (t, J = 5.0 Hz, 1H), 10.92 (s, 1H), 8.74 (s, 1H), 8.35 (s, 1H), 7.42 (s, 1H), 7.25 (s, 1H), 4.48 (t, J = 5.2 Hz, 1H), 3.70–3.59 (m, 2H), 3.49–3.40 (m, 2H), 2.56 (t, J = 7.5 Hz, 2H),

4.1.4. General synthetic procedure for 1a-b and 1j

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1.81–1.71 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H).

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Propargylamine (0.75 mmol) and AcOH (0.75 mmol) corresponding to a solution of compound 6a (138 mg, 0.5 mmol) in MeOH:DCE = 1:1 were added. The solution was stirred for 10 min, and then, NaBH3(CN) (62.8 mg, 1 mmol) was added. The mixture was stirred at room temperature for 12 h, and the solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using

4.1.4.1.

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mixtures of MeOH:CH2Cl2 as the eluent, obtaining the target compounds 1a-b and 1j. 1-Ethyl-3-(6-(4-((prop-2-yn-1-ylamino)methyl)-1H-imidazol-1-yl)

pyrimidin-4-yl)thiourea

(1a).

The

corresponding

propargylamine

was

EP

2-Propynylamine. White solid, 53% yield. Mp 166–167 °C; 1H NMR (400 MHz, CD3OD) δ 8.71 (d, J = 1.0 Hz, 1H), 8.55 (d, J = 1.2 Hz, 1H), 7.76 (s, 1H), 7.02 (d, J = 1.0 Hz, 1H), 3.86 (s, 2H), 3.74 (q, J = 7.3 Hz, 2H), 3.46 (d, J = 2.5 Hz, 2H), 2.67 (t, J

AC C

= 2.5 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H).

13

C NMR (125 MHz, DMSO-d6) δ 179.18,

159.95, 157.33, 154.44, 143.17, 134.64, 112.55, 94.29, 82.74, 73.79, 45.03, 39.69, 36.83, 13.65. HRMS (EI): m/z calcd C14H17N7S (M+) 315.1266, found 315.1269. HPLC purity: 98.0%.

4.1.4.2. 1-Ethyl-3-(6-(4-((methyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl) pyrimidin-4-yl)thiourea

(1b).

The

corresponding

propargylamine

was

N-methylpropargylamine. White solid, 30% yield. Mp 183–184 °C; 1H NMR (400 MHz, CD3OD) δ 8.71 (d, J = 1.0 Hz, 1H), 8.54 (d, J = 1.3 Hz, 1H), 7.78 (s, 1H), 7.02 (d, J = 1.0 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.65 (s, 2H), 3.39 (d, J = 2.4 Hz, 2H),

ACCEPTED MANUSCRIPT 2.71 (t, J = 2.4 Hz, 1H), 2.38 (s, 3H), 1.31 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 179.17, 159.94, 157.33, 154.39, 141.15, 134.70, 113.88, 94.46, 79.13, 75.84, 52.34, 44.53, 41.15, 39.69, 13.65. HRMS (EI): m/z calcd C15H19N7S (M+) 329.1423, found 329.1425. HPLC purity: 99.1%.

-1-yl)pyrimidin-4-yl)thiourea

(1j).

The

corresponding

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4.1.4.3. 1-Ethyl-3-(6-(4-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)-1H-imidazol propargylamine

was

1-(prop-2-yn-1-yl)piperazine. Yellow solid, 28% yield. Mp 160−162 °C; 1H NMR (400 MHz, CD3COCD3) δ 11.35 (br, 1H), 9.79 (s, 1H), 8.71 (s, 1H), 8.36 (d, J = 1.2

SC

Hz, 1H), 7.61 (s, 1H), 7.32 (s, 1H), 3.82–3.61 (m, 2H), 3.51 (s, 2H), 3.27 (d, J = 2.4 Hz, 2H), 2.69 (t, J = 2.4 Hz, 1H), 2.55 (br, 8H), 1.29 (t, J = 7.2 Hz, 3H).

13

C NMR

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(100 MHz, DMSO-d6) δ 179.18, 159.95, 157.34, 154.40, 140.94, 134.62, 113.86, 94.44, 79.36, 75.67, 54.95, 52.33, 51.05, 45.96, 39.73, 13.68. HRMS (EI): m/z calcd C18H24N8S (M+) 384.1845, found 384.1847. HPLC purity: 96.1%. 4.1.5. General synthetic procedure for 1c-i

To a solution of compound 1a (158 mg, 0.5 mmol) in CH3CN (8 mL),

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corresponding RX (0.55 mmol), Et3N (101 mg, 1 mmol), and a catalytic amount of KI were added. The reaction mixture was refluxed for 12 h, and the solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica gel

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column using mixtures of MeOH:CH2Cl2 as the eluent, affording the target compounds 1c-i.

4.1.5.1. 1-Ethyl-3-(6-(4-((ethyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)

AC C

pyrimidin-4-yl)thiourea (1c). Iodoethane was used as the corresponding RX. White solid, 27% yield. Mp 163−164 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.18 (br, 1H), 10.91 (s, 1H), 8.75 (s, 1H), 8.39 (s, 1H), 7.54 (s, 1H), 7.28 (s, 1H), 3.68–3.60 (m, 2H), 3.58 (s, 2H), 3.38 (s, 2H), 3.14 (s, 1H), 2.59–2.52 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.0 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 179.18, 159.95, 157.35,

154.37, 141.10, 134.75, 113.90, 94.43, 78.86, 75.77, 50.07, 46.45, 40.77, 39.73, 13.68, 12.47. HRMS (EI): m/z calcd C16H21N7S (M+) 343.1579, found 343.1583. HPLC purity: 98.0%. 4.1.5.2. 1-Ethyl-3-(6-(4-((prop-2-yn-1-yl(propyl)amino)methyl)-1H-imidazol-1-yl)

ACCEPTED MANUSCRIPT pyrimidin-4-yl)thiourea (1d). 1-Iodopropane was used as the corresponding RX. White solid, 30% yield. Mp 158−159 °C; 1H NMR (400 MHz, CD3OD) δ 8.71 (d, J = 1.0 Hz, 1H), 8.53 (d, J = 1.3 Hz, 1H), 7.75 (s, 1H), 7.02 (d, J = 1.0 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.69 (s, 2H), 3.43 (d, J = 2.4 Hz, 2H), 2.65 (t, J = 2.4 Hz, 1H),

3H).

13

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2.61–2.51 (m, 2H), 1.62–1.51 (m, 2H), 1.31 (t, J = 7.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, C NMR (125 MHz, DMSO-d6) δ 179.17, 159.92, 157.31, 154.36, 141.51,

134.65, 113.60, 94.38, 79.14, 75.51, 54.39, 50.37, 41.28, 39.69, 20.05, 13.63, 11.70. HRMS (EI): m/z calcd C17H23N7S (M+) 357.1736, found 357.1734. HPLC purity:

4.1.5.3.

SC

98.9%.

1-(6-(4-((Butyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)

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pyrimidin-4-yl)-3-ethylthiourea (1e). 1-Iodobutane was used as the corresponding RX. Faint yellow solid, 28% yield. Mp 112−114 °C; 1H NMR (400 MHz, CD3OD) δ 8.71 (d, J = 0.9 Hz, 1H), 8.53 (d, J = 1.2 Hz, 1H), 7.76 (s, 1H), 7.03 (d, J = 0.9 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.70 (s, 2H), 3.44 (d, J = 2.4 Hz, 2H), 2.66 (t, J = 2.4 Hz, 1H), 2.63–2.58 (m, 2H), 1.58–1.47 (m, 2H), 1.41–1.34 (m, 2H), 1.31 (t, J = 7.3 Hz, 13

C NMR (100 MHz, DMSO-d6) δ 179.18, 159.95,

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3H), 0.94 (t, J = 7.3 Hz, 3H).

157.34, 154.37, 141.44, 134.69, 113.68, 94.38, 79.08, 75.63, 52.07, 50.37, 41.29, 39.73, 28.97, 19.94, 13.88, 13.67. HRMS (EI): m/z calcd C18H25N7S (M+) 371.1892,

4.1.5.4.

EP

found 371.1895. HPLC purity: 96.6%.

N-((1-(6-(3-Ethylthioureido)pyrimidin-4-yl)-1H-imidazol-4-yl)

methyl)-N- (prop-2-yn-1-yl)acetamide (1f). Reagents used were compound 1a (158

AC C

mg, 0.5 mmol), acetyl chloride (47 mg, 0.75 mmol), Et3N (101 mg, 1 mmol), DCM (5 mL), rt, 12 h. Purification involved the use of MeOH/CH2Cl2 as eluent. Compound 1f: white solid, 45% yield. Mp 197−199 °C; 1H NMR (400 MHz, CD3OD) δ 8.73 (8.72) (d, J = 0.8 Hz, 1H), 8.58 (8.55) (d, J = 1.1 Hz, 1H), 7.88 (7.79) (s, 1H), 7.04 (7.02) (d, J = 0.8 Hz, 1H), 4.70 (4.65) (s, 2H), 4.25 (d, J = 2.4 Hz, 2H), 3.75 (q, J = 7.3 Hz, 2H), 2.81 (2.66) (t, J = 2.3 Hz, 1H), 2.30 (2.25) (s, 3H), 1.33 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 179.16, 169.65 (169.57), 159.94, 157.34, 154.31, 140.33 (139.86), 135.42 (134.84), 113.78 (113.63), 94.61 (94.46), 79.97 (79.61), 74.83 (73.86), 44.46 (41.92), 39.69 (s), 37.48 (33.55), 21.49 (21.38), 13.63. HRMS (ESI):

ACCEPTED MANUSCRIPT m/z calcd C16H19N7OS [M+H]+ 358.1405, found 358.1448. HPLC purity: 98.5%. 4.1.5.5.

1-(6-(4-((Benzyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)

pyrimidin-4-yl)-3-ethylthiourea (1g). (Bromomethyl)benzene was used as the corresponding RX. White solid, 48% yield. Mp 146−147 °C; 1H NMR (400 MHz,

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CD3OD) δ 8.71 (d, J = 0.9 Hz, 1H), 8.55 (d, J = 1.3 Hz, 1H), 7.78 (s, 1H), 7.40 (d, J = 7.0 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 0.9 Hz, 1H), 3.84–3.62 (m, 6H), 3.33 (s, 2H), 2.71 (t, J = 2.3 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 179.17, 159.92, 157.31, 154.34, 141.23, 138.51,

SC

134.79, 128.62, 128.23, 127.03, 113.80, 94.46, 78.77, 76.10, 56.60, 49.90, 41.05, 39.69, 13.64. HRMS (EI): m/z calcd C21H23N7S (M+) 405.1736, found 405.1737.

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HPLC purity: 98.0%.

4.1.5.6. 1-(6-(4-(((Cyanomethyl)(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl) pyrimidin-4-yl)-3-ethylthiourea

(1h).

2-Bromoacetonitrile

was

used

as

the

corresponding RX. Yellow solid, 36% yield. Mp 151−152 °C; 1H NMR (400 MHz, CD3OD) δ 8.72 (d, J = 0.9 Hz, 1H), 8.57 (d, J = 1.2 Hz, 1H), 7.84 (s, 1H), 7.02 (d, J =

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0.9 Hz, 1H), 3.82–3.77 (m, 4H), 3.74 (q, J = 7.3 Hz, 2H), 3.51 (d, J = 2.4 Hz, 2H), 2.79 (t, J = 2.4 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 179.16, 159.95, 157.35, 154.32, 139.49, 135.12, 116.01, 114.71, 94.56, 78.45, 76.67,

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49.67, 42.22, 40.77, 39.69, 13.64. HRMS (ESI): m/z calcd C16H18N8S [M+H]+ 355.1409, found 355.1454. HPLC purity: 97.1%. 4.1.5.7. 1-(6-(4-((Di(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl) pyrimidin

AC C

-4-yl)-3-ethylthiourea (1i). 3-Bromoprop-1-yne was used as the corresponding RX. White solid, 36% yield. Mp 151−153 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.17 (t, J = 5.2 Hz, 1H), 10.91 (s, 1H), 8.73 (s, 1H), 8.40 (s, 1H), 7.56 (s, 1H), 7.27 (s, 1H), 3.67 – 3.60 (m, 2H), 3.59 (s, 2H), 3.41 (d, J = 1.8 Hz, 4H), 3.20 (s, 2H), 1.22 (t, J = 7.2 Hz, 3H).13C NMR (100 MHz, DMSO-d6) δ 179.17, 159.96, 157.36, 154.35, 140.50, 134.93, 114.23, 94.52, 79.09, 75.90, 49.57, 41.28, 39.73, 13.69. HRMS (EI): m/z calcd C17H19N7S (M+) 353.1423, found 353.1402. HPLC purity: 98.9%. 4.1.6. General synthetic procedure for 7b-c PBr3 (541 mg, 2 mmol) was dropped to a solution of compounds 6b-c (2 mmol)

ACCEPTED MANUSCRIPT in DCM (30 mL). The mixture was stirred at room temperature overnight, and the solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using mixtures of MeOH:CH2Cl2 as the eluent to afford the target compounds 7b-c. 1-(6-(4-(2-Bromoethyl)-1H-imidazol-1-yl)pyrimidin-4-yl)-3-ethyl

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4.1.6.1.

thiourea (7b). White solid, 28% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.19 (t, J = 5.6 Hz, 1H), 10.98 (s, 1H), 8.75 (s, 1H), 8.40 (s, 1H), 7.61 (s, 1H), 7.27 (s, 1H), 3.76 (t, J = 6.9 Hz, 2H), 3.69–3.58 (m, 2H), 3.11 (t, J = 6.9 Hz, 2H), 1.22 (t, J = 7.2 Hz,

4.1.6.2.

SC

3H).

1-(6-(4-(3-Bromopropyl)-1H-imidazol-1-yl)pyrimidin-4-yl)-3-ethyl

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thiourea (7c). White solid, 30% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H), 11.03 (t, J = 5.4 Hz, 1H), 9.88 (s, 1H), 8.88 (s, 1H), 8.00 (d, J = 1.3 Hz, 1H), 7.46 (s, 1H), 4.36 (t, J = 7.3 Hz, 2H), 3.69–3.58 (m, 2H), 3.02 (t, J = 7.0 Hz, 2H), 2.63–2.54 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H).

4.1.7. General synthetic procedure for 2a-b and 3a-b

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A mixture of 7b-c (0.5 mmol) and K2CO3 (172 mg, 1.25 mmol) was stirred overnight in 2-propynylamine or N-methylpropargylamine (1 mL) at room temperature. The mixture was then evaporated to dryness under reduced pressure. The

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residue was purified on a silica gel column using mixtures of MeOH:CH2Cl2 as the eluent to afford the corresponding compounds 2a-b and 3a-b. 4.1.7.1.

1-Ethyl-3-(6-(4-(2-(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol-1-yl)

AC C

pyrimidin-4-yl)thiourea (2a). Yellow solid, 70% yield. Mp 159−160 °C; 1H NMR (400 MHz, CD3OD) δ 8.70 (d, J = 0.9 Hz, 1H), 8.52 (d, J = 1.3 Hz, 1H), 7.63 (s, 1H), 6.98 (d, J = 0.9 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.44 (d, J = 2.5 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 2.5 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H). 13

C NMR (100 MHz, DMSO-d6) δ 179.20, 159.95, 157.31, 154.44, 142.94, 134.47,

112.06, 94.17, 83.09, 73.53, 47.37, 39.73, 37.32, 28.04, 13.69. HRMS (EI): m/z calcd C15H19N7S (M+) 329.1423, found 329.1424. HPLC purity: 99.0%. 4.1.7.2.

1-Ethyl-3-(6-(4-(2-(methyl(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol

-1-yl)pyrimidin-4-yl) thiourea (2b). Yellow solid, 68% yield. Mp 162−164 °C; 1H

ACCEPTED MANUSCRIPT NMR (400 MHz, CD3OD) δ 8.70 (d, J = 0.9 Hz, 1H), 8.51 (d, J = 1.3 Hz, 1H), 7.65 (s, 1H), 6.98 (d, J = 0.9 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.44 (d, J = 2.4 Hz, 2H), 2.87–2.77 (m, 4H), 2.69 (t, J = 2.4 Hz, 1H), 2.40 (s, 3H), 1.31 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 179.19, 159.95, 157.33, 154.43, 142.66, 134.39,

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112.06, 94.19, 78.88, 75.88, 54.29, 44.83, 41.21, 39.73, 26.04, 13.68. HRMS (ESI): m/z calcd C16H21N7S [M+H]+ 344.1613, found 344.1657. HPLC purity: 99.0%. 4.1.7.3.

1-Ethyl-3-(6-(4-(3-(prop-2-yn-1-yl)amino)propyl)-1H-imidazol-1-yl)

pyrimidin-4-yl)thiourea (3a). Yellow solid, 65% yield. Mp 118−120 °C; 1H NMR

SC

(400 MHz, DMSO-d6) δ 11.19 (t, J = 5.3 Hz, 1H), 10.92 (s, 1H), 8.73 (s, 1H), 8.35 (d, J = 1.1 Hz, 1H), 7.41 (d, J = 15.7 Hz, 1H), 7.24 (s, 1H), 3.68–3.59 (m, 2H), 3.30 (d, J

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= 2.3 Hz, 2H), 3.03 (t, J = 2.3 Hz, 1H), 2.62–2.52 (m, 4H), 1.78–1.68 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 179.20, 159.96, 157.32, 154.45, 144.41, 134.53, 111.57, 94.17, 82.72, 73.79, 47.30, 39.73, 37.28, 28.28, 25.48, 13.69. HRMS (EI): m/z calcd C16H21N7S (M+) 343.1579, found 343.1573. HPLC purity: 98.3%.

1-Ethyl-3-(6-(4-(3-(methyl(prop-2-yn-1-yl)amino)propyl)-1H-imidazol

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4.1.7.4.

-1-yl)pyrimidin-4-yl) thiourea (3b). Yellow solid, 71% yield. Mp 122−124 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (t, J = 5.3 Hz, 1H), 10.92 (s, 1H), 8.74 (s, 1H),

EP

8.35 (d, J = 1.1 Hz, 1H), 7.44 (s, 1H), 7.24 (s, 1H), 3.68–3.60 (m, 2H), 3.30 (d, J = 2.3 Hz, 2H), 3.10 (t, J = 2.3 Hz, 1H), 2.56–2.52 (m, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.19 (s, 3H), 1.80–1.67 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)

AC C

δ 179.20, 159.93, 157.29, 154.45, 144.35, 134.50, 111.57, 94.17, 79.04, 75.63, 54.43, 44.92, 41.24, 39.69, 26.33, 25.41, 13.65. HRMS (EI): m/z calcd C17H23N7S (M+) 357.1736, found 357.1739. HPLC purity: 98.5%. 4.1.8. General synthetic procedure for 2d-c To a solution of compound 2a (164 mg, 0.5 mmol) in CH3CN (8 mL), corresponding RX (0.55 mmol), Et3N (101 mg, 1 mmol), and a catalytic amount of KI were added. The reaction mixture was refluxed for 12 h, and the solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using mixtures of MeOH:CH2Cl2 as the eluent, affording the target

ACCEPTED MANUSCRIPT compounds 2c-d. 4.1.8.1 1-Ethyl-3-(6-(4-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol-1-yl) pyrimidin-4-yl)thiourea (2c). Iodoethane was used as the corresponding RX. White solid, 33% yield. Mp 154−155 °C; 1H NMR (400 MHz, CD3OD) δ 8.72 (s, 1H), 8.54

RI PT

(s, 1H), 7.70 (s, 1H), 7.02 (s, 1H), 3.75 (q, J = 7.3 Hz, 2H), 3.71 (d, J = 1.8 Hz, 2H), 3.10 – 3.04 (m, 2H), 2.93 – 2.84 (m, 4H), 2.83 (s, 1H), 1.32 – 1.30 (m, 3H), 1.20 (t, J = 7.2 Hz, 3H).

13

C NMR (125 MHz, DMSO-d6) δ 179.18, 159.96, 157.35, 154.43,

134.49, 112.22, 94.22, 51.87, 46.93, 40.70, 40.11, 29.02, 13.68. HRMS (EI): m/z

4.1.8.2.

SC

calcd C17H23N7S (M+) 357.1736, found 357.1738. HPLC purity: 97.5%.

1-(6-(4-(2-((Cyanomethyl)(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol-1

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-yl)pyrimidin-4-yl)-3-ethylthiourea (2d). 2-Bromoacetonitrile was used as the corresponding RX. White solid, 35% yield. Mp 159−160°C; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (t, J = 5.3 Hz, 1H), 10.95 (s, 1H), 8.74 (s, 1H), 8.36 (d, J = 1.3 Hz, 1H), 7.52 (s, 1H), 7.25 (s, 1H), 3.84 (s, 2H), 3.68–3.59 (m, 2H), 3.45 (d, J = 2.4 Hz, 2H), 3.30 (t, J = 2.4 Hz, 1H), 2.86–2.80 (m, 2H), 2.77–2.71 (m, 2H), 1.22 (t, J = 7.2 13

C NMR (100 MHz, DMSO-d6) δ 179.20, 159.98, 157.37, 154.45, 141.97,

TE D

Hz, 3H).

134.58, 116.14, 112.34, 94.27, 78.43, 76.69, 51.74, 42.57, 41.21, 39.73, 25.70, 13.70. HRMS (EI): m/z calcd C17H20N8S (M+) 368.1532, found 368.1515. HPLC purity:

EP

95.8%.

4.2. Biological activity

4.2.1. In vitro inhibition of AChE and BuChE

AC C

The enzyme inhibition activity of the tested compounds was performed using the

method of Ellman et al. with slight modification [44]. The rat cortex was homogenized in cold 75 mM sodium phosphate buffer (pH 7.4) as the AChE source, and the rat serum was collected as the BuChE source. 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide and butyrylthiocholine iodide were purchased from Sigma. Huperzine A (Hup A) was used as reference compound. 1 µL of the tested compounds with an appropriate concentration (1 nM to 10 mM) was added into the assay solution, which consisted of 50 µL of 0.1 M phosphate buffer, 50 µL of 0.2% DTNB, 109 or 99 µL of deionized water, 10 µL of rat cortex homogenate or 20

ACCEPTED MANUSCRIPT µL of rat serum, and 30 µL of 2 mM acetylthiocholine iodide or 40 µL of 2 mM butyrylthiocholine iodide as the substrate of the AChE or BuChE enzymatic reaction, respectively. Then the mixture was incubated for 20 min at room temperature. The production of the yellow anion of 5-thio-2-nitrobenzoic acid was measured with a

RI PT

microplate reader (DTX 880, Beckman Coulter) at 450 nm. The inhibition percentage caused by the presence of test compound was calculated, and the IC50 was defined as the concentration of the compound that reduced 50% of the enzymatic activity without inhibitor.

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4.2.2. In vitro inhibition of hMAO-A and hMAO-B

MAO kit purchased from Invtrogen Company was used to evaluate the inhibitory

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activity for recombinant human MAOs of the target compounds. hMAO-A and hMAO-B (Sigma) were dilute to 12.5 µg/mL and 75 µg/mL using 1×buffer, respectively. Compounds were dissolved in DMSO (10 mM) and diluted in 50 mM KH2PO4 buffer (pH 7.4) to the desired final concentration. All the compounds are soluble at the tested concentration. Test drugs (50 µL) and MAOs (50 µL) were

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incubated at 37°C for 15 min in a 96-well costar plate. Amplex Red reagent working solution (100 µL), which consisted of 2 mM p-tyramine for hMAO-A or benzylamine for hMAO-B, 2 U/mL horseradish peroxide, and 200 mM Amplex Red reagent, was

EP

added into the mixture to start the reaction. After 15 min of incubation at 37 °C, the generated fluorescence was recorded by Molecular Devices (SpectraMax i3; excitation, 545 nm; emission, 590 nm). The specific fluorescence emission was

AC C

calculated after subtraction of the background activity. The background activity was determined from wells containing all components except the MAO isoforms, which were replaced by a sodium phosphate buffer solution. The percent inhibition was calculated by the following expression: Inhibitition% = [1－(Fi－Fblank) / (Fcontrol－Fblank)] × 100% Where Fi is the fluorescence of the compounds; Fcontrol is the fluorescence of the control group; Fblank is the fluorescence of background. 4.2.3. Oxygen radical absorbance capacity (ORAC-FL) assay

ACCEPTED MANUSCRIPT Compounds were dissolved in DMSO (10 mM) and diluted with 75 mM phosphate buffer (pH 7.4) to 20 µM. Fuorescein (FL) stock solution and 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) were prepared and diluted with 75 mM phosphate buffer (pH 7.4) to 0.117 µM and 40 mM, respectively. A solution of

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(±)-6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (Trolox) was diluted with the same buffer to 100, 80, 60, 50, 40, 20, and 10 µM. The mixture of the tested compounds (20 µL) and FL (120 µL) was incubated for 10 min at 37 °C, and then 60 µL of the AAPH solution was added. The fluorescence was recorded by Molecular

SC

Devices every two minutes for 180 min (SpectraMax i3; excitation, 485 nm; emission, 535 nm). A blank was determined using phosphate buffer instead of the tested

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compounds. All reaction mixtures were prepared in triplicate, and at least three independent runs were performed for each sample. The area under the fluorescence decay curve (AUC) after subtraction of the curve of the blank was calculated using

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the following equation:

In which f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. The net AUC was calculated by the expression AUCsample – AUCblank.

EP

The ORAC value for each sample were expressed as Trolox equivalents calculated from the ratio of the slopes of the concentration-response curves, the antioxidant vs Trolox.

AC C

4.2.4. Metal-chelating study The chelating studies were performed with a UV−vis spectrophotometer at

wavelengths ranging from 200 to 500 nm. The absorption spectra of compound 1b (40 µM, final concentration) alone or in the presence of CuCl2, FeSO4, FeCl3, or ZnCl2 (20 µM, final concentration) for 30 min in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded at room temperature. 4.2.5. Inhibition of ROS produced by copper redox cycling The tested compounds were dissolved in methanol and diluted with PBS buffer (20 mM, containing NaCl 100 mM, pH 7.4) to the desired final concentration. The

ACCEPTED MANUSCRIPT other solutions, except CuSO4 (dissolved in water and diluted in PBS), were mixed and diluted in the same PBS buffer with a final sample volume of 200 µL (ligand [15 µM] or PBS, Cu(II) [5 µM], deferoxamine [1 µM], CCA [50 µM], and then ascorbate [150 µM]). Each experiment was performed in triplicate. Hydroxyl radical production

RI PT

was measured as the conversion of CCA into 7-hydroxy-CCA by Molecular Devices at 37 °C (SpectraMax i3; excitation, 395 nm; emission, 450 nm). All of the test solutions contained 0.1% methanol. 4.2.6. Dot blot assay

SC

Aβ1−42 (Sigma, CA, U.S.) was dissolved in ammonium hydroxide (1% v/v) to afford a stock solution (2000 µM), and stored at −80 °C. For the experiments, the

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Aβ1−42 stock solution was diluted in 20 µM HEPES (pH 6.6) with 150 µM NaCl. The mixture of the peptide (25 µM, final concentration) with or without Cu(II) (25 µM, final concentration) and the tested compound (50 µM, final concentration) was incubated at 37 °C for 24 h. For the dot immunoblot assay, each Aβ sample was spotted (5 µL) on nitrocellulose membranes (GE Healthcare). Afterward, the

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membranes were blocked by 5% nonfat milk for 1 h and probed with A11 (Millipore) or 6E10 (Convance) antibodies at 4 °C overnight. The blots were washed with TBST and incubated with suitable horseradish peroxidase-conjugated secondary antibodies

EP

at room temperature for 1 h. Images were acquired by an image system (Tanon) and were analyzed using image J software. 4.2.7. TEM assay [50, 51].

AC C

In this experiment, experimental method was the same as the dot blot assay: The

Aβ1−42 stock solution was diluted with the same HEPES, and the mixture of Aβ (25 µM) solutions, ± CuCl2 (25 µM), ± compounds (50 µM) were incubated for 24 h at 37 °C. Aliquots (10 µL) of the samples were placed on a carbon-coated copper grid for 30 min. Each grid was stained with phosphotungstic acid (2%, 10 µL) for 20 s. After draining the excess staining solution, the specimen was transferred for imaging by transmission electron microscopy (JEM-1400). All compounds were solubilized in the buffer used for the experiment. 4.2.8. Cell viability assay

ACCEPTED MANUSCRIPT Primary cortical neurons (PCNs) were isolated from E17 SD rat embryos as described. Briefly, cortices were dissected in old high glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM, Invitrogen) and dissociated into single cells by trypsinization. Cells were seeded into 96-well plates coated with poly-L-lysine at a

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density of 3 × 104 cells per well. PCNs were cultured in a neurobasal medium (Invitrogen) containing 0.5 mM L-glutamine，2% B27 supplement, penicillin (60 mg/L) and streptomycin (50 mg/L). Half of the culture medium was refreshed every 3 days. The following experiments were performed at 9 days of culture [52]. For the

SC

neurotoxicity test, the cells were treated with compounds at 10 µM, 30 µM, and 100 µM for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,

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Sangon Biotech, China) analysis was performed to measure the cell viability. A total of 10 µL of MTT (5 mg/mL) was added into each well and incubated at 37 °C for 4 h. The production of MTT formazan was determined by measuring the absorbance at 490 nm. The cell viability was calculated and presented by the percentage of vehicle-treated group.

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For neuroprotection of compound 1b on Cu2+-induced Aβ1-42 neurotoxic model, the preparation of the Aβ1-42 sample was the same as that for the dot blot assay. The Aβ1-42 samples were added into the cell cultures at a concentration of 1 µM for 24 h.

vehicle group.

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Then, the cell viability was measured by the MTT assay and normalized by the

AC C

4.2.9. In vitro blood−brain barrier permeation assay Brain penetration of compounds was evaluated using a parallel artificial

membrane permeation assay (PAMPA), in a similar manner as described by Di et al [49]. Commercial drugs were purchased from Sigma and Alfa Aesar. The porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and the acceptor microplate were both from Millipore. The 96-well UV plate was from Corning Incorporated. The acceptor 96-well microplate was filled with 300 µL of PBS:EtOH (7:3, v:v), and the filter membrane was impregnated with 4 µL of PBL in dodecane (20 mg mL−1).

ACCEPTED MANUSCRIPT Compounds were dissolved in DMSO at 5 mg mL−1 and diluted to 100 µg mL−1 with PBS:EtOH (7:3, v:v), then 200 µL was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 12 h at 25 °C. After incubation, the donor plate was carefully removed

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and the concentration of compounds in the acceptor wells was determined using a UV plate reader (SpectraMax i3). Each sample was analyzed at eight wavelengths, in four wells, in at least three independent runs, and the results are given as the mean ±

SC

standard deviation. Pe was calculated using the following equation:

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where Vd is the volume in the donor well, Va is the volume in the acceptor well, A is the filter area, t is the permeation time, [drug]acceptor is the absorbance of the compound in the acceptor well, and [drug]equilibrium is the theoretical equilibrium absorbance.

In the experiment, 13 quality control standards of known BBB permeability were

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included to validate the analysis set (Table S1, Supporting Information). A plot of the experimental data versus reference values gave a strong linear correlation, Pe (exp.) = 0.974Pe (lit.) – 0.8152 (R2 = 0.9651) (Fig. S2, Supporting Information). From this equation and the limit established by Di et al. for blood−brain barrier permeation, we

EP

concluded that compounds with permeability greater than 3.08 × 10−6 cm s-1 could cross the blood−brain barrier and Pe values from 1.13×10−6 cm s-1 to 3.08 × 10−6 cm

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s-1 were classified as “CNS±” (BBB permeation uncertain) (Table S2, Supporting Information).

4.2.10. Cognitive performance evaluation Imprinting control region (ICR) mice weighing 18−25 g were obtained from the

Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). All procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as well as the guidelines of the Animal Care and Use Committee of Shanghai Institute of Materia Medica. The mice were trained to find the platform in a rectangular plastic channel water maze (80 cm ×

ACCEPTED MANUSCRIPT 50 cm × 20 cm), which was described in detail in our previous study [53]. Each mouse received at least two training sessions daily for four consecutive days before testing with the criterion of finding the platform within 30 s and committing less than three errors of entering no-exit paths. Mice that met the criterion were randomly

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divided into four treatment groups (N = 16): control (Ctrl, saline-treated) group, scopolamine (Scop) group, scopolamine (Scop) + huperzine A (Hup A) group, and scopolamine (Scop) + 1b·HCl group. To evaluate the cognitive-enhancing effect of compound 1b·HCl on scopolamine-induced cognitive impairment, scopolamine (4.5

SC

mg/kg) was injected (i.p.) 20 min before the behavior test, and compound 1b·HCl (30 mg/kg) or positive control (Hup A, 0.1 mg/kg) was administered (i.g.) 40 min prior to

recorded. 4.3. Molecular Docking Studies

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the administration of scopolamine. The escape latency and number of errors were

The simulation systems were built based on structures retrieved from the Protein Data Bank (PDB: 2V60 for hMAO-B, 5EIE for mAChE). For proteins, heteroatoms

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and water molecules were removed and hydrogen atoms were assigned using Protein Preparation Wizard. For ligand 1b, LigPrep was used to produce the structure of optimum energy. Docking simulations were performed using Glide in Schrödinger

EP

software [54].

Supplementary data

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Supplementary data include the synthesis of intermediate 4c, compound 1b·HCl, ThT fluorescence assay, raw data of the in vitro blood-brain barrier permeation assay and the NMR spectra of intermediates and final compounds.

Acknowledgments

Financial support for this research was provided by the National Natural Science Foundation of China (Grants 21672064, 21372001, 81522045), Yang-Fan Program from the Science and Technology Commission of Shanghai (Grants 17YF1403600), and the Fundamental Research Funds for the Central Universities.

Abbreviations used

ACCEPTED MANUSCRIPT AD, Alzheimer’s disease; AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; ACh, acetylcholine; AChEIs, AChE inhibitors; MAOs, monoamine oxidases; MAO-A, monoamine oxidase A; MAO-B, monoamine oxidase B; CAS catalytic active site; FAD, flavin adenine dinucleotide; Aβ, beta amyloid protein; BBB, blood−brain

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barrier; Hup A, huperzine A; CFDA, China Food and Drug Administration; ROS, reactive oxygen species; MTDLs, multitarget-directed ligands; ChEs, cholinesterases; SAR, structure activity relationship; rChE, rat cholinesterase; hMAO-A, human monoamine oxidase A; hMAO-B, human monoamine oxidase B; ORAC, free oxygen

SC

radical absorbance capacity; AAPH, 2,2'-azobis-(amidinopropane)dihydrochloride; EDTA, ethylenediaminetetraacetic acid; EGCG, epigallocatechin gallate; CCA,

ThT

thioflavine

T;

MTT,

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coumarin-3-carboxylic acid; CQ, Clioquinol; TEM, transmission electron microscopy; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide; PCN, primary cortical neuron; Don, donepezil; PAMPA, parallel artificial membrane permeation assay; mAChE, mice acetylcholinesterase; MP, melting point; TLC, thin-layer chromatography; LC-MS/MS, liquid chromatography tandem mass DTNB,

5,5′-dithiobis

(2-nitrobenzoic

acid);

HEPES,

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spectrometry;

2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid; PBS, phosphate buffer salt. PBL, porcine brain lipid.

EP

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•

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•

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•